X-Message-Number: 31105
Date: Wed, 8 Oct 2008 09:44:37 -0700 (PDT)
From: 
Subject: The Cryonics Institute Makes Another Technical Disclosure II

Biotechnol Lett. 2005 May;27(9):655-60.

Protection of osteoblastic cells from freeze/thaw cycle-induced oxidative stress
by green tea polyphenol.

    Han DW, Kim HH, Lee MH, Baek HS, Lee KY, Hyon SH, Park JC. Department of 
    Medical Engineering, Yonsei University College of Medicine, 134 
    Shinchon-dong, Seodaemun-gu, Seoul, 120-752, Korea.

    Green tea polyphenol (GTP) together with dimethylsulphoxide (DMSO) were 
    added to a freezing solution of osteoblastic cells (rat calvarial 
    osteoblasts and human osteosarcoma cells) exposed to repeated freeze/thaw 
    cycles (FTC) to induce oxidative stress. When cells were subjected to 3 
    FTCs, freezing medium containing 10% (v/v) DMSO and 500 mug GTP ml(-1) 
    significantly (p<0.05) suppressed cell detachment and growth inhibition by 
    over 63% and protected cell morphology. Furthermore, the alkaline 
    phosphatase activity of osteoblastic cells was appreciably maintained after 
    2 and 3 FTCs in this mixture. Polyphenols may thus be of use as a cell 
    cryopreservant and be advantageous in such fields as cell transplantation 
    and tissue engineering.
PMID: 15977073

J Neurosci Methods. 2005 Jun 30;145(1-2):255-66.

Optimal conditions for peripheral nerve storage in green tea polyphenol: an 
experimental study in animals.

    Matsumoto T, Kakinoki R, Ikeguchi R, Hyon SH, Nakamura T. Department of 
    Orthopedic Surgery, Graduate School of Medicine, Kyoto University, Sakyo-ku,
    Kyoto 606-8507, Japan.

    Our previous study demonstrated successful peripheral nerve storage for 1 
    month using polyphenol solution. We here report two studies to solve 
    residual problems in using polyphenols as a storage solution for peripheral 
    nerves. Study 1 was designed to determine the optimal concentration of the 
    polyphenol solution and the optimal immersion period for nerve storage. Rat 
    sciatic nerve segments were immersed in polyphenol solution at three 
    different concentrations (2.5, 1.0, and 0.5 mg/ml) for three different 
    periods (1, 7, and 26 days). Electrophysiological and morphological studies 
    demonstrated that nerve regeneration from nerve segments that had been 
    immersed in 1mg/ml polyphenol solution for 1 week and in Dulbecco's modified
    Eagle's medium (DMEM) for the subsequent 3 weeks was superior to the 
    regeneration in other treatment groups. In study 2, the permeability of 
    nerve tissue to polyphenol solution was investigated using canine sciatic 
    nerve segments stored in 1.0mg/ml polyphenol solution for 1 week and in DMEM
    for the subsequent 3 weeks. Electron microscopy revealed that the Schwann 
    cell structure within 500-700 microm of the perineurium was preserved, but 
    cells deeper than 500-700 microm were badly damaged or had disappeared. The 
    infiltration limit for polyphenol solution into neural tissue is inferred to
    be 500-700 microm.
PMID: 15922041

Kidney Int. 2003 Feb;63(2):554-63.

Bioflavonoids attenuate renal proximal tubular cell injury during cold 
preservation in Euro-Collins and University of Wisconsin solutions.

    Ahlenstiel T, Burkhardt G, Kohler H, Kuhlmann MK. Department of Medicine, 
    Division of Nephrology and Hypertension, University Hospital of Saarland, 
    Homburg/Saar, Germany.

    BACKGROUND: Cold ischemia and reperfusion during kidney transplantation are 
    associated with release of free oxygen radicals and damage of renal tubular 
    cells. Bioflavonoids may diminish cold storage-induced injury due to 
    antioxidant and iron chelating activities. This study was designed to 
    delineate the renoprotective mechanisms of bioflavonoids and to define the 
    structural features conferring cytoprotection from cold injury. METHODS: 
    LLC-PK1 cells were preincubated for three hours with bioflavonoids and cold 
    stored in University of Wisconsin (UW)- or Euro-Collins (EC)-solution for 20
    hours. After rewarming, cell viability was assessed by the lactate 
    dehydrogenase (LDH) release, MTT-test, and amino acid transport activity. 
    Lipid peroxidation was assessed from the generation of thiobarbituric 
    acid-reactive substances. RESULTS: Twenty-hours of cold storage of LLC-PK1 
    cells resulted in a substantial loss of cell integrity that was more 
    pronounced in the EC (LDH release, 93.6 +/- 1.6%) than the UW solution (67.2
    +/- 6.9%; P < 0.0001). Pretreatment with quercetin significantly enhanced 
    cell survival (LDH release, 5.4 +/- 2.7% for UW and 8.4 +/- 4.2% for EC) in 
    a concentration dependent manner. Structure-activity studies revealed 
    similar renoprotection for kaempferol, luteolin and fisetin, unlike 
    myricetin, morin, apigenin, naringenin, catechin, silibinin and rutin. Lipid
    peroxidation was reduced (UW alone, 2.7 +/- 1.2 vs. UW+quercetin 0.5 +/- 
    0.2 nmol/mg protein, P < 0.01), and l-threonine uptake completely sustained 
    by pretreatment with quercetin, kaempferol, luteolin, and fisetin. However, 
    renoprotection by fisetin was rapidly lost during rewarming. Protective 
    properties of bioflavonoids were governed by the number and arrangement of 
    hydroxyl substitutes, electron-delocalization, sterical planarity, and 
    lipophilicity of the basic diphenylpyran skeleton. CONCLUSION: Cold 
    storage-induced renal tubular cell injury is ameliorated by bioflavonoids. 
    Renoprotective effects of bioflavonoids are defined by structure, suggesting
    that flavonoids are incorporated into membrane lipid bilayers and interfere
    with membrane lipid peroxidation.
PMID: 12631120

J Biosci Bioeng. 2003;96(6):559-63.

Novel function of rare catechin, epigallocatechin-3-(3''-O-methyl)gallate, 
against cold injury in primary rat hepatocytes.

    Kagaya N, Hara Y, Saijo R, Kamiyoshi A, Tagawa Y, Kawase M, Yagi K. Graduate
    School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita,
    Osaka 565-0871, Japan.

    Epigallocatechin-3-(3''-O-methyl)gallate (EGCg-3''-OMe) is a rare component 
    in green tea leaf and its bioactivity is hardly known. In this paper, we 
    report that EGCg-3''-OMe has the function for cold preservation of primary 
    rat hepatocytes. Confluent primary cultured hepatocytes were suspended in a 
    storage solution, culture medium or cell banker (CB). EGCg-3''-OMe was 
    tested as a supplement in the storage solution together with a general 
    cryoprotectant, dimethylsulfoxide (DMSO). After 24 h cold preservation of 
    cells at 4 degrees C followed by 1 h rewarming, cell viability and 
    urea-synthesizing activity, one of the most important liver functions, were 
    measured. EGCg-3''-OMe dose-dependently maintained cell viability and this 
    effect was equal to that of a commercial CB at the highest concentration. 
    Cell viability was also maintained after a further 24 h incubation at 37 
    degrees C of the cold-preserved hepatocytes. Conversely, urea-synthesizing 
    activity was dose-dependently reduced by EGCg-3''-OMe. Cell protection by 
    EGCg-3''-OMe due to the decrease in metabolic activity in cold-preserved 
    cells was suggested. The decreased hepatic function of cells caused by 
    EGCg-3''-OMe was rescued after a further 24 h incubation of cells at 37 
    degrees C.
PMID: 16233573

Biol Pharm Bull. 2002 Sep;25(9):1156-60.

Enhancing effect of zinc on hepatoprotectivity of epigallocatechin gallate in 
isolated rat hepatocytes.

    Kagaya N, Kawase M, Maeda H, Tagawa Y, Nagashima H, Ohmori H, Yagi K. 
    Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan.

    The influence of metal ions (Fe2+, Cu2+, Zn2+) on the hepatoprotective 
    activity of epigallocatechin gallate (EGCG) against hepatotoxin-induced cell
    injury was investigated. Primary cultures of rat hepatocytes were treated 
    with a well-known hepatotoxin, bromobenzene (BB), in the presence of EGCG 
    only or EGCG plus each metal ion. After 24 h, 0.02 mM EGCG did not show 
    protective activity on the cultured hepatocytes. In contrast, the 
    hepatocytes were protected against BB in the presence of 0.02 mM EGCG and 
    0.02 mM zinc. The addition of only zinc could not protect hepatocytes 
    against BB. These results suggest that the formation of the zinc-EGCG 
    complex is very important in the enhancement of the hepatoprotective 
    activity of EGCG. The complexation of EGCG with zinc was confirmed by UV-VIS
    absorption spectroscopy.
PMID: 12230108

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